Telomere length is a parameter of interest not only with respect to the study of the telomere biology but also as a marker for aging and cancer. Regarding aging, it is known that telomere length decreases with age because telomerase activity in adult tissue is not sufficient to prevent telomere shortening, thus compromising cellular viability (Harley et al., 1990 and Blasco et al., 1997). In the case of cancer cells, telomere length is maintained due to the over-expression of telomerase or due to the activation of alternative mechanisms which promote telomerase elongation (Kim et al., 1994 and Bryan et al., 1997).
Thus, telomere length can be used both in aging studies, as a marker of biological fitness of human populations (Cawthon et al., 2003; Epel et al., 2004, and Valdes et al., 2005, Lancet, 366:662-664), in cancer and in screening methods for the identification of compounds interfering with said biological fitness.
The most widely used method for determining telomeric length is the so-called telomere restriction fragment assay (Moyzis et al., 1988). This method is based on a Southern blot hybridisation of a telomeric restriction fragments derived from genomic DNA using probes specific for the telomere repeats. However, TRF is a time-consuming technique which requires plenty of cells and only provides an average telomeric length of the cell population under study without giving an indication of telomere length in individual cells.
Another method for determining telomere length is quantitative fluorescent in situ hybridisation (FISH) based on the use of fluorescence microscopy on a preparation of metaphasic cells using a telomere-specific probe (Lansdorp et al., 1996; Zijlmans et al., 1997), (Martens et al., 1998). This technique is also cumbersome and time-consuming and requires cells in metaphase, which excludes from all those cells which can not proliferate in culture.
Another method for determining telomere length is flow fluorescent in situ hybridisation (flow-FISH) based on the determination of telomeric fluorescence in interphase cells using flow cytometry wherein the cells are labelled with a fluorescently-labelled telomere-specific probe (Rufer et al., 1998; Baerlocher et al., 2006). However, this method is only applicable to cells in suspension and the results are frequently biased due to auto-fluorescence of the cytoplasm.
The hybridization protection assay described by Nakamura et al., (Clinical Chemistry, 1999, 45:1718-1724) is based on a chemoluminescence determination of the amount of telomere-specific probe and normalized to the signal obtained with an Alu repeat-specific probe. However, this method requires a constant number of Alu repeats in the genome.
Other methods for determining fluorescence length include the hybridization assay (Freulet-Marriere et al, 2004), primed in situ labeling (PRIMS) (Therkelsen et al., 1995), PCR-based methods such as STELA (Baird, D. M., et al., 2003) and quantitative PCR (Cawthon, R. M., 2002).
The identification of adult stem cell compartments is essential for studying adult stem cell properties and regulation, as well as for their potential use in regenerative medicine.
The common approach to locate stem cell niches has been based on the different expression of a protein marker, or more usually a complex set of protein markers, in stem cell environments compared to more differentiated compartments, as well as on the general property that stem cells are long-term residents of a tissue and have a low proliferative rate (i.e. label-retaining techniques) (Fuchs et al., 2004, Cell, 116:769-778; Moore and Lemischka, 2006, Science, 311:1880-1885). These approaches are limited because each type of stem cell niche has its own specific set of markers.
Cotsarelis et al. (Cell, 1990, 61:1329-1337), Potten et al., (Int. J. Exp. Pathol., 1997, 78: 219-243) and Zhang et al. (Nature, 2003, 425:836-841) have relied on the identification of long-term retention cells (LRCs) for the identification of skin, intestinal, and hematopoietic stem cells. This assay is based in the identification, using DNA labeling, of cells in a given tissue that undergo slow cycling as measured by their ability to retain the labeled DNA for a much longer period than the rapid cycling progenitor cells. However, it is still not completely undisputed that LRCs are stem cells (Kiel et al. Nature, 2007, 449:238-42).
Doetsch et al., (Cell, 1999, 97:703-716), Ohlstein and Spradling, (Nature, 2006, 439:470-474), Palmer et al., (Mol. Cell Neurosci., 1997, 8:389-404) and Sanai et al., (Nature, 2004, 427:740-744) have used in vivo lineage tracing to search for cells that give rise to the downstream lineages to identify neural stem cells and Drosophila gut stem cells were identified.
Kim et al., (Cell, 2005, 121:823-835) have relied on the identification of multipotent cells, as revealed by their co-expression of multiple downstream lineage markers of stem cells. In this way, lung stem cells were identified as bronchioalveolar stem cells (BASCs) based on their co-expression of two downstream Clara and Alveolar lineage markers, CCA and SP-C, and their ability to give rise to Clara and Alveolar lineages.
The ability of stem cells to express certain types of multiple drug-resistant genes and display a unique pattern in flow cytometry assay has also been used to identify the so-called side population (SP). SP has been shown to be enriched with HSCs (Goodell et al., 1997, Nature Medicine, 3:1337-1345) and stem cells in other non-hematopoietic tissues (Goodell et al., Methods Mol. Biol., 2005, 290:343-352).
Additionally, several methods for the identification of adult stem cells have been developed based on functional characteristics of stem cells such as binding to soybean agglutinin (Reisner et al., 1982, Blood 59:360-363), resistance to the treatment of either 5-fluorouracil (Gordon et al., 1985, Leukemia research 9:1017-1021 and Berardi et al., Science 1995267:104-108) or alkylating agent (Sharkis et al., 1997, Stem cells (Dayton, Ohio) 15 Suppl 1, 41-44; discussion 44-45) and density-gradient (Juopperi et al., 2007, Experimental hematology, 35:335-341).
WO07124125 describes a method for the identification of stem cells wherein a cell population is treated with a DNA damaging agent, which results in the quiescent stem cells residing on the tissue become activated in order to replenish lost cells. These cells can be detected using a marker of DNA biosynthesis.
Thus, there is a need in the art for additional methods for the determination of telomere length and for methods for the identification of stem cell compartments within adult tissues which overcome the disadvantages of the methods known in the art as well as which are generally applicable to any tissue.